Dietary selenium levels modulates antioxidant,cytokine and immune response and selenoproteins mRNA expression in rats under heat stress condition

BackgroundHigher environmental temperature is a major abiotic stress factor for animals and human beings. The selenium (Se) is an important trace mineral having diverse health promoting effects under stress conditions. However, studies on dietary requirement of selenium under prolonged heat stress condition are lacking. Present study discern the effect of higher dietary Se levels on antioxidant, cytokine, haemato-biochemical profile, and immune response, and the selenoproteins mRNA expression in rats under prolonged heat stress (HS) condition.MethodsWeaned Wistar rats (4 wk age; 67.6 ± 1.53 g BW; n = 72) housed under thermoneutral (TN) or HS conditions and fed with purified diets containing three graded Se levels were divided in six experimental groups. The groups were 1) TN control with 138 ppb Se (TN_CON), 2) HS control with 138 ppb Se (HS_CON), 3) TN with higher Se @ 291 ppb (TN_Se1), 4) HS with higher Se @ 291 ppb (HS_Se1) 5) TN with higher Se @ 460 ppb (TN_Se2), 6) HS with higher Se @ 460 ppb (HS_Se2). Rats in all the six groups were maintained in TN environmental conditions (57.3 ± 0.22 temperature humidity index; THI) for initial 28 days period. Subsequently, rats of HS groups were exposed to 77.0 ± 0.11 THI for 6 h/d in a psychrometric chamber for last fourteen days.ResultsHigher dietary Se (291 and 460 ppb) significantly improved the blood hemoglobin concentration and reduced serum alanine aminotransferase activity of rats under HS conditions. The serum triiodothyronine and insulin levels were significantly higher in high dietary Se groups irrespective of the environmental conditions. Similarly, the serum reduced glutathione levels, and catalase and superoxide dismutase enzyme activity were increased and malondialdehyde levels were reduced in high dietary Se groups irrespective of stress conditions. The glutathione peroxidase (GPx) activity was significantly higher in 460 ppb dietary Se groups as compared to other groups. The serum pro-inflammatory cytokine interleukin (IL)− 1 was declined, whereas the anti-inflammatory cytokine IL-10 level was increased in high dietary Se fed rats under both HS and TN conditions with 460 ppb dietary Se groups showing pronounced effects. Further, there was heat stress- and dietary Se level dependent- up regulation in hepatic GPx and iodothyronine deiodinase-II mRNA expression and similar pattern was noticed in hepatic thioredoxin reductase mRNA expression. The selenoprotein-P mRNA expression was up regulated in 460 ppb Se fed HS group as compared to CON and Se1_C groups. High dietary Se improved the humoral immune response 7d after antigen inoculation under HS conditions whereas cell-mediated immune response was augmented in rats fed higher Se under TN condition.ConclusionIt is concluded that under prolonged heat stress conditions the dietary requirement of Se may be increased to 460 ppb for improving the antioxidant status and humoral immune response, cytokine levels, modulating the thyroid and insulin hormone, and the selenoproteins mRNA expression of rats.     

Physical habitat of zebra mussel (Dreissena polymorpha) in the lower Ebro River (Northeastern Spain): influence of hydraulic parameters in their distribution

Dreissena polymorpha (zebra mussel) is a freshwater bivalve mollusc and has been present for more than one decade in Spain. The zebra mussel causes serious ecological and socioeconomic impacts in areas where they settle. Our research aims to analyze its hydraulic habitat, developing physical models for this species, which indicates its preference and optimal microhabitat requirements. To get it, a survey was completed in a lotic reach in the lower Ebro River (Tarragona, Spain). Habitat suitability curves for the variables include water velocity, depth, Froude number, velocity/depth ratio, shear stress and shear stress ratio (RSS). In addition, interactive effects between hydraulic parameters on habitat selection and its use were studied and bivariate habitat models were developed. A close relationship was observed between the D. polymorpha presence and mean flow velocity combined with depth, Froude number and RSS. Suitable habitat requirements for Dreissena were detected in areas with stable river beds under high flows, and velocity below 1.2 m/s with a depth of less than 5 m under regular flows. Information about zebra mussel preferences may enhance the design of monitoring programmes and the integrated control management of this invasive mussel.     

蔗糖添加对杉木低磷胁迫响应和蔗糖代谢的影响

为了揭示低磷胁迫下蔗糖对杉木低磷胁迫响应和蔗糖代谢的影响,选用两种不同磷效率杉木家系M32和M28进行低磷胁迫下的蔗糖添加试验,分析蔗糖添加对低磷胁迫下杉木形态特征、生理特性和低磷诱导相关基因表达的影响。结果表明：蔗糖添加促进了低磷胁迫下杉木苗高、根长、根表面积、根平均直径、根体积、根叶组织蔗糖含量和根叶组织无机磷含量的增加,但仍明显低于正常供磷处理下添加蔗糖处理的杉木增量。低磷促进杉木叶中花青素的积累,而正常供磷和低磷胁迫下的蔗糖添加处理都显著促进了叶片花青素含量的增加。随着胁迫时间的延长,M28与M32在根、叶组织的蔗糖含量存在显著差异,且M28根叶组织中的蔗糖合成酶活性和蔗糖磷酸合成酶活性都高于M32。蔗糖合成酶ClSuSy在M28和M32根系中受低磷胁迫诱导下调表达,但蔗糖添加处理明显诱导ClSuSy表达量升高,M28在正常供磷并添加蔗糖处理下的ClSuSy表达量显著高于其它处理。蔗糖转运蛋白SUT4、磷转运蛋白ClPht1;4、紫色酸性磷酸酶PAP1和PAP11在M28和M32根系中总体上受低磷胁迫诱导上调表达,且受蔗糖添加处理诱导下调表达。低磷胁迫下,添加或不添加蔗糖处理的M32根系SUT4的表达量均在15d时显著升高,并在45d时回落到正常水平。ClPht1;4和PAP1在低磷胁迫15d的表达量显著高于45d时的表达量,且ClPht1;4在M32根系中的表达量远高于M28。本研究表明,蔗糖对杉木低磷胁迫响应和糖代谢有重要的影响作用,低磷胁迫下添加蔗糖处理能够在一定程度上缓解杉木低磷胁迫响应。     

Effects of temperature stress on NO-synthase and tyrosine hydroxylase activities in the central nervous system of bivalve molluscs

Effects of temperature stress on activities of NO-synthase (NOS) and tyrosine hydroxylase (TH) in the CNS of two species of bivalve molluscs, Mizuchopecten yessoensis and Chlamys farreri nipponensis (Pectinidae) were studied using NADPH-diaphorase histochemistry and immunocytochemistry. General and specific peculiarities in distribution and relative proportion of TH- and NO-containing neurons in the CNS nerve ganglia were revealed in norm and under stress at 30°C for 10, 30, and 60 min. The initial stress stage (for 10 min) has been found to be accompanied by an increase of the relative content of TH-positive neurons in some CNS areas of both mollusc species. In intact Chlamys farreri nipponensis, the presence of NOS in the CNS and its significant activation under temperature stress might have possibly been an important neuroprotective component of stress reaction in some mollusc species.     

饥饿对草鱼非特异免疫水平的影响

研究了长期饥饿对草鱼(Ctenopharyngodon idellus)非特异性免疫水平的影响。实验选取平均体质量(31.86±1.47)g的草鱼,随机分为2个实验组(对照组和饥饿组),每组3个平行,饥饿处理15、30、45和60 d,测定饥饿对草鱼头肾和脾中自然杀伤(NK)细胞的杀伤活性、血清和肝胰脏中溶菌酶活性、血清中碱性磷酸酶活性的影响。结果表明:受饥饿胁迫的影响,草鱼自然杀伤性细胞在脾和头肾中的杀伤活性显著低于对照组(P0.05)且不随着饥饿时间的延长发生显著性变化;随着饥饿时间的延长,血清和肝胰脏中溶菌酶呈现先升高后降低的趋势,血清碱性磷酸酶在饥饿15、45、60 d时显著低于对照组;饥饿组的碱性磷酸酶活性在饥饿30 d以后,维持恒定。由此可见,长时间的饥饿胁迫降低了草鱼的免疫水平。相比较而言,自然杀伤细胞的杀伤活性在反映鱼类免疫状况时比溶菌酶和碱性磷酸酶可能更为灵敏。     

Physiological and metabolic adjustments of Hoplosternum littorale (Teleostei,Callichthyidae) during starvation

All animals face the possibility of limitations in food resources that could ultimately lead to mortality caused by starvation. The primary goal of this study was to characterize the various physiological strategies that allow fish to survive starvation. A multiparametric approach, including morphological biomarkers, blood plasma metabolites, oxidative stress and energy reserves, was used to assess starvation effects on the fish Hoplosternum littorale. Adult specimens were maintained at four experimental groups: control (fed ad libitum), and starved (not fed) fish for 7 and 28 days. Significant changes were observed not only after 28 days, but also after 7 days of starvation. In the shorter period, the hepatosomatic index as well as plasma triglycerides and glucose were significantly lower in starved fish than in the control ones. These results were accompanied by reduced lipid, glycogen and protein reserves in liver and diminished glycogen content in muscle, suggesting the need of these macromolecules as fuel sources. In addition, increased antioxidant enzyme activities were observed in gills, without evidence of oxidative stress in any of the evaluated tissues. Most significant differences were found in 28-days starved fish: total body weight together with the hepatosomatic index was lower when compared to control fish. The plasmatic metabolites tested (glucose, triglyceride, cholesterol and protein), all energy reserves in liver and glycogen content in muscle decreased in 28-days starved fish. Lipid oxidative damage was reported in liver, kidney and brain, and antioxidant enzymes (GST, GR, GPx and CAT) were activated in gills. According to the multivariate analysis, oxidative stress markers and metabolic parameters were key biomarkers that contributed in separating starved from fed fish. Our study allowed an integrated assessment of the fish response to this particular condition.     

Evaluation of phagocytic activity and nitric oxide generation by molluscan haemocytes as biomarkers of inorganic arsenic exposure

The natural habitats of the freshwater bivalve Lamellidens marginalis face the risk of contamination by the toxic metalloid arsenic. Haemocyte-mediated non-self phagocytosis and generation of nitric oxide (NO) as reactive nitrogen intermediate were examined to establish the reliability of the parameters as biomarkers of sodium arsenite-induced stress on the animal at sublethal concentrations. The studies suggest imposition of a remarkable immune compromise/immune suppression on the animal by the natural contaminant. The animal expressed partial recovery in its phagocytic potential and NO generation over a period of 30 days. Quantitation of phagocytic efficiency and intrahaemocyte NO generation indicates the possibility of the parameters be accepted as cellular biomarkers to estimate and characterize the vulnerability of the freshwater organisms to sodium arsenite-induced stress.     

饥饿胁迫对草鱼鱼种非特异免疫力的影响

研究长期饥饿对草鱼(Ctenopharyngodon idellus)鱼种非特异性免疫力的影响。实验选取平均体质量(31.86?1.47g)的草鱼,随机分为2个实验组(对照组和饥饿组),每组三个平行,饥饿处理15、30、45和60d,测定饥饿对头肾和脾脏中自然杀伤(NK)细胞的杀伤活性、血清和肝胰脏中溶菌酶活性、血清中碱性磷酸酶活性的影响。结果表明：受饥饿胁迫的影响,草鱼鱼种自然杀伤性细胞在脾脏和头肾中的杀伤活性显著低于对照组(P<0.05)且不随着饥饿时间的延长发生显著性变化;随着饥饿时间的延长,血清和肝胰脏中溶菌酶呈现先升高后降低的趋势,血清碱性磷酸酶在饥饿15d、45d、60d时显著低于对照组;,饥饿组的碱性磷酸酶活性在饥饿30d以后,维持恒定。由此可见,长时间的饥饿胁迫降低了草鱼鱼种的免疫力。相比较而言,自然杀伤细胞的杀伤活性在评价鱼类的免疫状况时比溶菌酶和碱性磷酸酶更为灵敏。     

Effects of starvation and - or -alanine administration on the free - and -alanine levels in the muscle and hepatopancreas of the crayfish, Procambarus clarkii

Changes of - and -alanine and other free amino acids were examined in muscle and hepatopancreas of the crayfish Procambarus clarkii before and after a 1-month starvation in freshwater, 50% seawater, and seawater. Total free amino acids and - and -alanine in both tissues decreased during starvation. The largest decrease was found in the animals starved in freshwater. In 50% seawater and full-strength seawater, the ratio of -alanine to total alanine increased in both tissues, although the level was reduced a little. A large dose of -alanine administered into the tail muscle of freshwater crayfish was converted to -alanine and -alanine returned to the control level within a week. A large amount of -alanine was transported from muscle to hepatopancreas. -Alanine injected into freshwater crayfish was converted to -alanine, but was not transported to the hepatopancreas. In the case of -alanine injection into the muscle of crayfish acclimated to 50% seawater, large amounts of - and -alanine were maintained in the muscle during the experimental period of 4 days. These data indicate that -alanine is metabolized actively in crayfish tissues and accumulated as an important osmolyte during osmotic stress.     

Localization of NO-Synthase in Gonads of the Freshwater Bivalve Mollusc Nodularia vladivostokensis

Ultrastructural localization of NO-synthase in the gonad of the freshwater bivalve mollusc Nodularia vladivostokensis was studied. Individual NO-containing nerve fibers are revealed in the gonad wall. In the interacinar connective tissue, a high level of the enzyme activity is found in neurons and neural fibers. Reaction product in neurons is located in the nucleus, granular endoplasmic reticulum, on plasma membrane, and in the area of neuromuscular synapses. NO-containing granules are found in various nerve processes, but only cholinergic ones are identified.     

Genome-Wide Analysis of DNA Methylation in Five Tissues of Zhikong Scallop,Chlamys farreri

DNA methylation plays a vital role in tissue development and differentiation in eukaryotes. Epigenetic studies have been seldom conducted in the extremely diverse and evolutionarily highly successful bilaterian lineage Mollusca. In the present study, we conducted the genome-wide profiling of DNA methylation for five tissues of a bivalve mollusc, Chlamys farreri using the methylation-sensitive amplification polymorphism (MSAP) technique. The methylation levels were quite similar among tissues, ranging from 20.9% to 21.7%. CG methylation was the dominant type (14.9%–16.5%) in the C. farreri genome, but CHG methylation also accounted for a substantial fraction of total methylation (5.1%–6.3%). Relatively high methylation diversity was observed within tissues. Methylation differentiation between tissues was evaluated and 460 tissue-specific epiloci were identified. Kidney differs from the other tissues in DNA methylation profiles. Our study presents the first look at the tissue-specific DNA methylation patterns in a bivalve mollusc and represents an initial step towards understanding of epigenetic regulatory mechanism underlying tissue development and differentiation in bivalves.     

The effects of feeding condition and dietary lipid level on lipoprotein lipase gene expression in liver and visceral adipose tissue of red sea bream Pagrus major

The effects of feeding condition and dietary lipid level on lipoprotein lipase (LPL) gene expression in the liver and visceral adipose tissue of red sea bream Pagrus major were investigated by competitive polymerase chain reaction. Not only visceral adipose tissue but also liver of red sea bream showed substantial LPL gene expression. In the liver, starvation (at 48 h post-feeding) drastically stimulated LPL gene expression in the fish-fed low lipid diet, but had no effect in the fish fed high lipid diet. Dietary lipid level did not significantly affect the liver LPL mRNA level under fed condition (at 5 h post-feeding). In the visceral adipose tissue, LPL mRNA number per tissue weight was significantly higher in the fed condition than in the starved condition, irrespective of the dietary lipid levels. Dietary lipid levels did not affect the visceral adipose tissue LPL mRNA levels under fed or starved conditions. Our results demonstrate that both feeding conditions and dietary lipid levels alter the liver LPL mRNA levels, while only the feeding conditions but not dietary lipid levels cause changes in the visceral adipose LPL mRNA level. It was concluded that the liver and visceral adipose LPL gene expression of red sea bream seems to be regulated in a tissue-specific fashion by the nutritional state.     

Microalgae triacylglycerols content by FT-IR spectroscopy

The present study aims to develop a methodology via Fourier transform infrared (FT-IR) spectroscopy for the semiquantitative determination of triacylglycerols (TAGs) in microalgal consortia, consistent with the use of the technique as process control. FT-IR spectroscopy has proved to be a powerful analytical tool for the identification of macromolecular pools (e.g., proteins, lipids, and carbohydrates) and in monitoring biochemical changes (including lipids) in response to nutrient stress or environmental modifications. In the Oocystis-based consortium under examination, the synthesis of neutral lipid in the form of TAGs can be induced, applying stress condition, and these lipids are suitable as biodiesel precursors. In the exponential growing phase, the consortium shows a low TAGs content, in the order of 5 %w, that can be increased till around 22 %w on ash free dry matter, after nitrogen starvation.     

Adequate dietary protein level enhances stress resistance and immune status of blunt snout bream (Megalobrama amblycephala Yih, 1955)

This study hypothesized that an optimum dietary protein level might play an important role in improving stress tolerance, enhancing an immune function, and ultimately minimizing temperature stress. For this purpose, the present study conducted a 10‐week feeding trial followed by a 7‐day stress experiment to evaluate the effects of dietary protein levels (28%–36%) on the physiological performances and hepatic Hsp70 gene expression in blunt snout bream Megalobrama amblycephala fry under a high temperature challenge. Fish fry (initial weight, 16.08 ± 0.03 g, n = 25) were fed with their respective diets to apparent satiation, and samples taken once before stress and four times during high temperature stress days (0.125, 0.5, 2 and 7 days). Serum total protein and cholesterol contents before stress were affected by dietary protein levels; during stress these parameters showed no significant changes up until day 2, showing some changes thereafter. Regardless of 0.125, 0.5, 2 or 7 days during stress, cortisol and aspartate aminotransferase levels in the group fed 32% dietary protein were significantly lower than in the other groups. At 0.125, 0.5 or 2 days during stress, the complement component 3 (C3) and 4 (C4) levels increased significantly up to certain levels, declining thereafter. The expression level of Hsp70 mRNA before stress was not affected by dietary protein levels, but increased significantly from 0.125 to 0.5 days during stress, and was reduced thereafter. Overall, low cortisol and high C3, C4 and Hsp70 mRNA levels were found in the group fed 32% protein throughout the stress experiment, which indicated that an adequate protein level enhances stress resistance and immunity in fish.     

淡水贝类贝壳多层构造形成研究

对几种淡水贝（包括蚌、螺）进行形态及组织学观察，并通过实验方法重现贝壳三种物质，即：角质、棱柱质、珍珠质的生成过程，结果表明：外套膜外表皮细胞是由相同类型细胞组成，这些相同细胞在不同的作用条件下形成贝壳多层构造。     

Effects of thiols and blockers of sulfhydryl groups on negative regulation of the adenylyl cyclase signaling system by biogenic amines

Effect of a thiol-containing compound, β-mercaptoethanol, and of blockers of cysteine sulfhydryl groups (para-chlormercury benzoate andN-ethylmaleimide) on hormonal negative regulation of functional activity of the adenylyl cyclase system was studied in smooth muscles of the freshwater bivalve molluscAnodonta cygnea (agonist of ß-adrenoreceptors—isoproterenol) and in the rat skeletal muscles (serotonin). It was found that in the presence of ß-mercaptoethanol the inhibitory effects of isoproterenol (mollusc) and serotonin (rat) were preserved. At the same time, the blockers of SH-groups disturb essentially both the hormonal effects and their potentiation in the presence of Gpp[NH]p, this process being practically irreversible in the presence ofN-ethylmaleimide. Thus, it has been shown that the negative hormonal regulation in the muscle membranes of the mollusc and rat is dependent on the SH-group state in the protein components of the hormonesensitive adenylyl cyclase system.     

Effect of hydrogen peroxide on antioxidant enzymes and metallothionein level in the digestive gland of Mytilus galloprovincialis. cavalett@unipmn.it

The pro-oxidant effect of H2O2 at a concentration of 20 microM was examined in the digestive gland of Mytilus galloprovincialis, a bivalve mollusc frequently used in biomonitoring programs. The oxidative stress caused by H2O2 has been evaluated in terms of lipid peroxidation and lysosomal system alteration. Complex cellular antioxidant defence mechanisms of the mussel were investigated at the enzymatic and non-enzymatic level in order to explain their relative role in reducing the risk of oxidative injury. Metallothionein, glutathione, superoxide dismutase, catalase and glutathione peroxidase were assayed after 1, 4 and 7 days of exposure to H2O2. The metallothionein content showed an increase by 43% after 4 days of exposure, followed by a decrease back to control values at 7 days. Antioxidant enzyme activities followed a similar pattern with a moderate increase after 1 or 4 days of treatment and a return to control values at 7 days. All data indicate a 'transient' oxidative stress response, after which mussel cells restore the redox balance.     

Molecular characterization of AwSox2 from bivalve Anodonta woodiana: Elucidating its player in the immune response

Sox2 is an embryonal stem cell Ag essential for early embryonic development, tissue homeostasis and immune regulation. In the current study, one complete Sox2 cDNA sequence was cloned from freshwater bivalve Anodonta woodiana and named AwSox2. Histological changes of testis derived from Bisphenol A (BPA) treatment were analyzed by hematoxylin and eosin staining. Expressions of AwSox2 derived from BPA, LPS and polyinosinic:polycytidylic (Poly I:C) challenge were measured by quantitative real-time PCR. The full-length cDNA of AwSox2 contained an open reading frame of 927 nucleotides bearing the typical structural features of Sox2 family. Obvious degeneration, irregular arrangement of spermatids, and clotted dead and intertwined spermatids were observed in BPA-treated groups. Administration of BPA could result in a dose-dependent up-regulation of AwSox2 expression in the male gonadal tissue of A. woodiana. In addition, expression of AwSox2 was significantly induced by LPS and Poly I:C treatment in the hepatopancreas, gill and hemocytes, compared with that of control group. These results indicated that up-regulations of AwSOx2 are closely related to apoptosis of spermatogonial stem cells derived from BPA treatment as well as enhancement of immune defense against LPS and Poly I:C challenge in A. woodiana.     

The effects of starvation and refeeding on plasma amino acid levels in carp, Cyprinus carpio L., 1758

Plasma amino acid (AA) levels of carp, Cyprinus carpio L., 1758, were analysed after various periods of starvation as well as after 12 days of refeeding. The levels were compared to control groups, which had been previously fasted for 24 h. A positive correlation between dietary and plasma essential amino acid (EAA) concentrations was observed in all of the control groups.
The effect of starvation on the dynamics of AA concentration was different according to the period of starvation. Fasting already produced a decrease of total α-AA levels at 2 days, and these low levels were maintained until 5 and 8 days. These short periods of fasting affected the levels of EAA (especially branched-chain AA) more than those of non-essential amino acids (NEAA). The only AA that increased was Ala, which rose at 5 days of starvation, surpassing the levels of the control group. These high levels were maintained until 19 days of starvation.
A different situation was observed at 19 days, when an important increase of total α-AA levels was produced, the branched-chain AA being the most notable among EAA and Glu/Gln among NEAA. Later, at 50 days of starvation, total α-AA, EAA (except Leu and Ile) and NEAA decreased.
After 50 days of starvation, 12 days of refeeding did not modify the levels of EAA and NEAA, and their concentrations were lower than those of the control group, which presented an increase of total AA at this time. The differences observed on the changes of individual AA levels are discussed.     

The influence of the landscape structure within buffer zones,catchment land use and instream environmental variables on mollusc communities in a medium-sized lowland river

The world’s freshwater molluscan fauna is facing unprecedented threats from habitat loss and degradation. Declines in native populations are mostly attributed to the human impact, which results in reduced water quality. The objectives of our survey were to analyse the structure of the mollusc communities in a medium-sized lowland river and to determine the most important environmental variables at different spatial scales, including landscape structure, catchment land use and instream environmental factors that influence their structure. Our survey showed that a medium-sized river, that flows through areas included in the European Ecological Natura 2000 Network Programme of protected sites, provides diverse instream habitats and niches that support 47 mollusc species including Unio crassus, a bivalve of Community interest, whose conservation requires the designation of a special conservation area under the Habitats Directive Natura 2000. This survey showed that mollusc communities are impacted by several environmental variables that act together at multiple scales. The landscape structure within buffer zones, catchment land use and instream environmental variables were all important and influenced the structure of mollusc communities. Therefore, they should all be taken into consideration in the future restoration of the river, future management projects and programmes for the conservation of biodiversity in running waters. The results of this study may be directly applicable for the rehabilitation of river ecosystems and are recommended to stakeholders in their future decision concerning landscape planning, monitoring species and their habitats, conservation plans and management in accordance with the requirements of sustainable development.